Precision modeling of mitochondrial disease in rats via DdCBE-mediated mtDNA editing

Mitochondrial DNA (mtDNA) mutations manifest mainly as base changes, resulting in a broad spectrum of serious diseases in human 1 . To date, more than 270 pathogenic variants of mtDNA in humans have been reported and the number continues to rise 2 . Currently, no curative treatments for patients with mtDNA pathogenic mutations are available. There is an urgent need for the generation of animal models harboring precise human mtDNA variants to reveal the physiopathology and develop therapeutic approaches for these diseases. Mammalian animal models with pathogenetic mtDNA mutations could be generated by mitochondrial transplantation or screening new mtDNA mutations by using PolG D257A/WT lineages 3 , whereas neither of these strategies could produce animals with precise mtDNA mutations on demand. Recently, a bacterial toxin deaminase (DddA) from Burkholderia cenocepacia , was reported to be able to convert cytosine to uracil specifically within dsDNA. The toxin domain of DddA (DddA tox ,1264–1427 amino acids) could be engineered and incorporated with mitoTALE system to efficiently achieve C ∙ G-to-T ∙ A conversion in mtDNA of human cell lines 4 . This breakthrough technology, DddA-derived cytosine base editor (DdCBE), paves a new way to produce animal models with desired mtDNA mutations. Lately, DdCBE is used to mediate mtDNA editing in mouse, but does not cause apparent phenotype 5 . Rat is an important and widely used laboratory model and has many advantages, especially in physiology, toxicology, and pharmacology study. In this study, we explored the application of DdCBE in rats to generate a mitochondrial disease model with pathogenetic mtDNA mutations.